PP3 forms stable tetrameric structures through hydrophobic interactions via the C‐terminal amphipathic helix and undergoes reversible thermal dissociation and denaturation

Abstract

The milk protein proteose peptone component 3 (PP3), also called lactophorin, is a small phosphoglycoprotein that is expressed exclusively in lactating mammary tissue. The C-terminal part of the protein contains an amphipathic helix, which, upon proteolytic liberation, shows antibacterial activity. Previous studies indicate that PP3 forms multimeric structures and inhibits lipolysis in milk. PP3 is the principal component of the proteose peptone fraction of milk. This fraction is obtained by heating and acidifying skimmed milk, and in the dairy industry milk products are also typically exposed to treatments such as pasteurization, which potentially could result in irreversible denaturation and inactivation of bioactive components. We show here, by the use of CD, that PP3 undergoes reversible thermal denaturation and that the a-helical structure of PP3 remains stable even at gastric pH levels. This suggests that the secondary structure survives treatment during the purification and possibly some of the industrial processing of milk. Finally, asymmetric flow field-flow fractionation and multi-angle light scattering reveal that PP3 forms a rather stable tetrameric complex, which dissociates and unfolds in guanidinium chloride. The cooperative unfolding of PP3 was completely removed by the surfactant n-dodecyl-b-D-maltoside and by oleic acid. We interpret this to mean that the PP3 monomers associate through hydrophobic interactions via the hydrophobic surface of the amphipathic helix. These observations suggest that PP3 tetramers act as reservoirs of PP3 molecules, which in the monomeric state may stabilize the milk fat globule. Structured digital abstractl PP3 and PP3 bind by circular dichroism (View interaction) l PP3 and PP3 bind by molecular sieving (View interaction) l PP3 and PP3 bind by fluorescence technology (View interaction) l PP3 and PP3 bind by molecular sieving (View interaction) Abbreviations AF4, asymmetric flow field-flow fractionation; DDM, n-dodecyl-b-D-maltoside; DOPC, 1,2-dioleoylphosphatidylcholine; DOPG, 1,2-dioleoylphosphatidylglycerol; GdmCl, guanidinium chloride; MALS, multi-angle light scattering; MRE, mean residue ellipticity; OA, oleic acid; PP3, proteose peptone component 3; RI, refractive index; SEC, size exclusion chromatography; TFE, trifluoroethanol.

Effect of fermentation temperature on the properties of exopolysaccharides and the acid gelation behavior for milk fermented by Streptococcus thermophilus strains DGCC7785 and St-143

“…The PP3 preferentially remains in multimeric structures of 4 to 10 times higher molar mass than its monomeric form. The molar mass of isolated multimeric PP3 fraction were reported to be 1.9 −2.3 × 10 5 g/mol (Sørensen et al, 1997;Pedersen et al, 2012), which were similar to the molar mass of the protein fraction co-isolated with EPS in our study (Figure 5).…”

“…The most abundant contaminant, PP3, also called lactophorin, or glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1), is one of the components of the proteose peptone fraction of milk. Bovine milk contains about 1 g/L of proteose peptone fraction, including ~25% (200-300 mg/L) PP3 (Pedersen et al, 2012). The PP3 is composed of 135 AA residues and is highly soluble in water, as it is highly glycosylated.…”

“…The PP3 has 3 O-glycosylation sites at 34(T), 78(S), and 104(T) residues, 1 N-glycosylation site at 95 (N) residue, and 5 phosphoserine residues at 47, 52, 56, 58, and 64 positions in the sequence. The theoretical molar mass of PP3 is 1.72 × 10 4 g/mol; however, its total molar mass of the monomeric form was reported to be higher (1.93-2.8 × 10 4 g/mol) due to the presence of glycans attached at different glycosylation sites (Coddeville et al, 1998;Pedersen et al, 2012). The PP3 preferentially remains in multimeric structures of 4 to 10 times higher molar mass than its monomeric form.…”

Effect of Total Proteosepeptone Content on the Variability of Bovine Milk Foaming Property

“…Hence, the slow foaming attitude of M1 samples could be related to the higher presence of PP in which, probably, the PP3 component was not in prevalence. PP3 is expressed exclusively in mammary tissue of ruminants and it is not formed after the milk ejection (Pedersen et al, 2011). Several studies showed that bovine PP3 is a good substrate for PL that reduces the lactophorin to a shorter fragment (residue 54-135) that has lost the emulsifying properties (Sørensen and Petersen, 1993).…”

Genotype-by-environment interaction in Holstein heifer fertility traits using single-step genomic reaction norm models

“…RAD51 has been reported to be associated with bovine oocytes meiosis progress [49,50]. Some candidate genes (e.g., LAP3, GLYCAM1, PDE1B, MICALL2, NPC1) are related with other economically-important traits in cattle such as milk production, carcass traits, body weight, body height, and body length [51][52][53][54][55]. Additionally, several genes were annotated to be associated with stress response in cattle.…”